Comparative evaluation of the effects of three hydraulic calcium silicate cements on odontoblastic differentiation of human dental pulp stem cells: an in vitro study

Abstract Objective The study aimed to compare the response of human dental pulp stem cells (hDPSCs) towards three hydraulic calcium silicate cements (HCSCs) by measuring cytotoxicity and expression of dentinogenic genes. Methodology Dental pulps of five impacted mandibular third molars were extirpated as a source for hDPSCs. Next to culturing, hDPSCs were subjected to fluorescence-activated cell sorting after the third passage to validate stemness of the cells. Human DPSCs were exposed to diluted supernatants of OrthoMTA (OMTA), Biodentine (BD) and Calcium-Enriched Mixture (CEM) at concentrations 10, 25, 50 and 100% at the first, third and fifth day of culture. Then, cells were exposed to 10% concentrations supernatant of HCSCs to determine DSPP and DMP1 gene expression, using a quantitative polymerase-chain reaction. Data were analyzed using one-way and three-way ANOVA, followed by Tukey post hoc statistical tests. Results Optimal cell proliferation was observed in all groups, regardless of concentration and time-point. HCSC supernatants were non-cytotoxic to hDPSCs at all three time-points, except for 100% Biodentine on day five. On day seven, OMTA group significantly upregulated the expression of DSPP and DMP1 genes. On day 14, expression of DMP1 and DSPP genes were significantly higher in BD and OMTA groups, respectively. Conclusion Biodentine significantly upregulated DMP1 gene expression over 14 days, whereas CEM was associated with only minimal expression of DSPP and DMP1 .


Introduction
Vital pulp treatments (VPT) are used to preserve pulpal integrity and function in immature permanent teeth. However, recent studies have focused on VPT use in mature teeth with carious pulpal exposures with partial irreversible pulpitis. 1 Following successful VPTs, such as direct pulp capping (DPC), deposition and organization of hydroxyapatite crystals take place in the dentine-pulp complex, regulated by a collagen template and non-collagenous acidic proteins. Hydraulic calcium silicate cements (HCSCs) are used widely in VPT due to their impact on the formation of calcified bridges over exposed pulps. 2 Upon proper choice of material in VPT, pulpal space is successfully sealed to hamper bacterial penetration and maintain remnant pulpal health following the formation of a calcified dentine-like bridge. 1 Therefore, presence of viable human dental pulp stem cells (hDPSCs) enables a yielded respond, migration, proliferation and, eventually, differentiation into secretory cells for formation of this bridge. 3 The hDPSCs can be also applied in regenerative endodontic treatments (RETs), whose stem cells should remain viable after being exposed to HCSCs directly or indirectly to continue root formation. 4 HDPSCs reside in specific niches and remain in their undifferentiated state as a reservoir within their microenvironment. 5 Viable hDPSCs can differentiate into secretory odontoblast-like cells via regulative impacts of signaling molecules such as transforming growth factor-beta (TGF-β) superfamily including TGF-β1 and -3, fibroblast growth factor (FGF), bone morphogenetic proteins (BMPs) such as BMP-2, BMP-4, BMP-7, and heme oxygenase-1 enzyme. 5 The PI3K/AKT/mTOR signaling pathway modulates the proliferation of hDPSCs in a sequential fashion, mediated by growth factors, cytokines and environmental stimuli. 5 Members of the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family such as dentin matrix protein-1 (DMP1), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), bone sialoprotein (BSP), and dentine sialophosphoprotein (DSPP) are associated with dentine bio-mineralization. 6 The hDPSCs, which can be detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence, can represent markers of odontoblastic differentiation, including those of the noncollagenous acidic proteins. Dentine sialophosphoprotein (DSPP) is present in bone, dentine, cementum and, to a much lower extent, in some non-mineralized tissues.
The DSPP gene expression in dentine is greater than that in non-dental tissues. The cleaved products of DSPP gene are dentine phosphoprotein (DPP) and dentine sialoprotein (DSP), resulting in formation of active fragments from an inactive precursor that allows spatiotemporally controlled mineralization of dentine. 7 DPP is a chief promoter of the biomineralization process via hydroxyapatite (HA) crystals formation. This is assumed to result from established electrical affinity between negatively charged portions of DPP and positively charged calcium, making it easier for Ca ++ ions to be presented to collagen fibers located in the mineralization front. 7 Abundant in peritubular dentine, DMP1 is another member of the SIBLING family and an important component of the non-collagenous dentine matrix. 6 During normal and pathological dentine formation, DMP1 can induce differentiation of hDPSCs into odontoblastlike cells and regulate dentine mineralization and organization of the collagenous matrix. 8    A standardized 0.2 cm 3 increment of each cement was transferred to a 12-well plate using minimal pressure to homogenously cover the bottom of each well. The samples were incubated for 24 h at 37ºC and 5% CO2 with 100% humidity, and sterilized by gamma irradiation with an intensity of 37 kGy, activity of 6820 Ci and dose rate of 1.62 Gy/sec. Finally, 3 mL high-glucose DMEM was added and incubated at 37ºC and 5% CO2 with 100% humidity for another 24 h. The samples were prepared so that the exposed cement surface to liquid ratio was established at 126.6 mm 2 /mL according to ISO standards. 21 After 24 h, the supernatant was removed and filtered using a 0.45-size filter and 10, 25, 50 and 100% concentrations were used for the subsequent cellular viability tests to determine the most appropriate dilution for the real-time polymerase chain reaction (RT-PCR) (n=3).

Cell proliferation assay
Initially, 10 4 cells were counted using hemocytometer and added to 48-well plates with 500 µL of the standard culture medium for 24 h of incubation. Afterwards, the supernatants of the test materials with 10, 25, and 50% dilutions were added to the plates and incubated  Table 1.

Results of MTT assay
Based on the one-way ANOVA test, we observed an insignificant difference in cell proliferation after exposure to 10, 25, 50, and 100% concentrations OMTA and CEM cement in first, third, and fifth days (p>0.05).
Furthermore, we identified an insignificant difference in cell proliferation following exposure to different concentrations of BD in first and third days (p>0.05).
However, the test presents a significant decrease in cell proliferation, following exposure to 100% concentration of BD (p=0.012). Three-way ANOVA test showed that cell proliferation of test materials in different concentrations had an insignificant difference in days 1 and 3 compared to control group (p>0.05).
Contrarily, when exposed to 100% concentration of BD cell proliferation was significantly lower than control on day five (p=0.016) (Figure 4).

Results on day seven
We verified a significant difference in DSPP and DMP1 genes expression between genes expression between test materials ( Figure 2) on day seven. The complementary Tukey HSD test revealed that OMTA significantly increased expression of the aforementioned genes (p=0.0002) ( Figure 5).

Results on day 14
One-way ANOVA test indicated a significant difference in expression of DSPP and DMP1 genes between test materials on day 14. The Tukey HSD test indicated that Biodentine significantly increased DMP1 expression (p<0.001) and OMTA significantly increased DSPP gene expression (p=0.0003) ( Figure 6).  Figure 4-MTT assay. The one-way ANOVA test showed no significant difference in cell proliferation after exposure to 10, 25, 50, and 100% concentrations OMTA and CEM cement in days one, three and five. (p>0.05) Furthermore, no significant difference was observed in cell proliferation following exposure to different concentrations of BD in first and third days (p>0.05). The test demonstrated a significant decrease in cell proliferation following exposure to 100% concentration of BD (p=0.012). Three-way ANOVA test showed that cell proliferation of test materials in different concentrations presented no significant difference in days one and three compared to control group (p>0.05). On the other hand, cell proliferation was significantly lower than control on day five when exposed to 100% concentration of BD (p=0.016) The similar cyto-compatibility of CEM and MTA using hDPSCs has been reported. 25 Although the detailed information about the composition of the MTA used in that study was suppressed, the authors reported an initial decrease in cell viability, which was not observed in this study. We can attribute this to the direct material- In this study, OMTA had greater expression of DSPP and DMP1 genes compared to CEM and positive and negative control groups. However, Asgary, et al. 34 (2014) reported that the expression of DSPP and DMP1 showed no significant difference between and among MTA, CEM and differentiation medium, although the expression was higher than growth medium used as the control.

Regarding cytotoxicity, cell proliferation and
This can be attributable to the difference in exposure of the test material with the cells. Presumably, direct contact of CEM could result in its improved odontogenic profile.
The effect of calcium silicate-based materials on hDPSCs has been evaluated frequently, increasing the conclusions due to different constituents of the materials tested; methodologic details such as diversities in assessments; acquisition of material extracts; direct versus indirect contact of the test material with cellular medium, etc. In this study, we used 10% dilution of the test material extracts for evaluation of DSPP and DMP1 gene expressions.
If cells had direct contact with experimental cements, as stated by Asgary,et al. 34 (2014)